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Journal of Clinical and Translational Research 2015; 1(3): 190-204
Journal of Clinical and Translational Research
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ORIGINAL ARTICLE
How the vagus nerve produces beat-to-beat heart rate variability;
experiments in rabbits to mimic in vivo vagal patterns
John M. Karemaker
Department of Anatomy, Embryology and Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
ABSTRACT
A RT I C L E I N F O
Article history:
Received: 17 November, 2015
Revised: 6 December, 2015
Accepted: 19 December, 2015
Published online: 20 December, 2015
Keywords:
sinoatrial node
vagotomy
phase response curve
respiratory sinus arrhythmia
baroreflex
cardiac autonomic plexus
cardiac baroreflex
Background and Aim: Analysis of heart rate variability (HRV) has recently become the playing field of
mathematicians and physicists, losing its relation to physiology and the clinic. To set the record straight, a
set of animal experiments is presented here, which was designed to test how vagus nerve traffic might
produce beat to beat (b-t-b) heart rate (HR) control, like the baroreflex will do in vivo.
Methods: The response of HR to vagus nerve stimulation was tested after bilateral vagotomy in rabbits
under anesthesia. Three protocols were followed: 1. Single burst stimulation at varying moments in one
cardiac cycle; 2. B-t-b stimulation in each cycle, coupled to the P-wave with variable delays; in addition,
testing the effects of one increased or decreased burst; 3. Tetanic stimulation, shortly interrupted or increased at varying moments in the cardiac cycle.
Results and Conclusions: Sensitivity of the sinoatrial node to the timing of vagal bursts in its cycle from
protocol 1 explains most of the observations. A single burst would be most effective when applied in late
repolarization or early diastole of the sinoatrial node’s action potential. In b-t-b stimulation the longest
cardiac cycles occur when bursts are timed just before the end of the ‘sensitive period’. Later coming bursts
have their (diminished) effect on the next cycle; critically timed bursts induce an unstable HR, alternating
between long and short cycles. This ran in synchrony with the respirator, thus producing a large respiratory
sinus arrhythmia, even though the vagus nerves had been cut. HR-response to vagal burst activity shows
two components: a fast one which is phase-sensitive and a slow one that builds up with longer lasting activity and also disappears slowly. Tetanic stimulation results in prolonged, but variable cycle lengths which are
difficult to change by short-lasting manipulation of impulse frequency, be it up or down.
Relevance for patients: Measurement of heart rate variability (HRV) and baroreflex sensitivity (BRS) have
become clinical tools in the cardiology clinic and in hypertension research. This study shows how the underlying vagus nerve to heart rate physiology is responsible for moment-to-moment variability in these
numbers at almost unchanged underlying physiology. Programmed stimulation of the vagus nerves in acute
animals (rabbits) demonstrates that the optimal mode of fast, beat-to-beat heart rate control by these nerves
is by means of bursts of impulses arriving in every heart beat at well-timed moments. In vivo this is how
the baroreflex stabilizes blood pressure at the expense of HRV.
1. Introduction
Heart rate variability is, boldly put, the price paid by the
blood pressure control system to obtain blood pressure stability.
Figure 1 demonstrates this hypothesis schematically: the blood
pressure upstroke induces baroreceptor afferent impulses at
each heartbeat, which are immediately turned into vagus nerve
efferent activity to slow down the sinoatrial node. If, for instance, the heart produces a larger stroke volume (the second
beat in the schema) this is sensed by the baroreceptors and
*Corresponding author:
John M. Karemaker
Systems Physiology, Academic Medical Center, P.O. 22660, University of Amsterdam, 1100 DD Amsterdam, the Netherlands
Tel: +31 20 5664827
E-mail: [email protected]
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Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
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Figure 1. Blood pressure control by the fast (vagally mediated) baroreflex. Top trace blood pressure, next schematic baroreceptors afferent signal, (ECG
for time reference), then cardiac vagus nerve efferent activity, delaying the sinus node’s depolarization (lower trace). In the second beat systolic blood
pressure is increased (red line) by an increased stroke volume; the baroreceptors react by more afferent impulses, resulting in more efferent vagal activity,
which is delaying the upcoming beat by longer hyperpolarizing the sinus node. Consequently, the next diastolic pressure is not as much increased as the
grey lines suggests but already more or less stabilized. Figure adapted from [2] and [3].
transformed via the vagus nerve into more slowing down.
Consequently, the next diastolic pressure is already more or
less stabilized, depending on the effectiveness of the baroreflex. This idea on how the vagus nerve helps control blood
pressure is the center piece of the ‘DeBoer’ model [1] which
helps to understand the relationship between heart rate and
blood pressure variability in daily life.
Heart rate variability (HRV) is also a reflection of the autonomic nervous system: sympathetic and parasympathetic (vagus nerve) activity, and circulating hormones. The fast
beat-to-beat changes can be attributed to changes in vagal activity. This makes HRV the ideal candidate for non-invasive
and almost continuous observation of a person’s autonomic
condition: little HRV and higher heart rates would imply sympathetic (over-)activity, lower heart rates with large beat-tobeat changes imply tranquility with parasympathetic domination. This maxim has ruled the field of HRV-analysis for a long
period of time. Recently it was challenged from 3 angles: 1)
the observation that over a long range of heart rates in different
species HR and HRV are strongly related, leading to the notion
that HRV is just another way of looking at HR [4,5], combined
with: 2) New analysis of sinoatrial node physiology, stressing
that the pacemaking process itself is subject to inherent variability, due to the properties of the tissue and the surrounding
micro-milieu [6]. 3) The heart has integrating nervous centers
of its own in the cardiac ganglionic plexuses, where integration of autonomic influences and instantaneous cardiac demands take place [7]. In the present paper a synthesis of the
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old and new views is attempted, based on vagus nervesinoatrial node physiology in vivo. For this purpose, an old set
of animal experiments has been re-analyzed. Earlier, the results had only been presented orally, focused on the oscillator
properties of the sinoatrial node [8]. Recently a partial result of
the re-analysis has been presented as abstract [9].
Two modes of activity have been observed in efferent cardiac vagal traffic [10-12]: in pulsatile mode there are bursts of
impulses in each cardiac cycle, reflexly coupled to the upstroke of the pulse wave, sensed by baroreceptive afferent areas in the circulation; in tetanic mode, vagal activity is more or
less continuous, loosely coupled to the level of blood pressure,
but also to chemoreceptor- and other afferent nerve traffic. In
the present experimental animal study both modes of efferent
cardiac vagal activity have been tested for their aptitude to
induce changes in heart rate (HR) from one beat to the next.
In the literature a large number of papers can be found on
heart rate responses to vagal stimulation, as reviewed in [13].
However, although short-lasting bursts of stimuli and repeated
bursts have been tested for their ability to pace and synchronize heart rate, as in [14] no studies are available that looked at
variations in burst-amplitude (i.e. number of applied stimuli)
per heart beat as mode of heart rate regulation.
When it comes to the question of how fast heart rate can
react to the vagus, a classical approach is to systematically
shift a strong burst of vagal activity through the cardiac cycle
and measure its effect on the ongoing and following cycle durations (Figure 2a). Since the timing of the vagal burst does
not only decide which cycle is influenced - the ongoing or the
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Karemaker | Journal of Clinical and Translational Research 2015;1(3): 190-204
Figure 3b). Such experiments have been done here as well.
When considering the resulting curves, a number of questions
came up:
1. Will the PRC change when heart rate is lowered by concurrent vagal stimulation, to mimic a changed vagal tone?
2. Is there a PRC for short-lasting decreases of vagal activity, i.e. for an increase in heart rate? Of course, this can only be
measured when a constant level of background vagal activity
is generated.
3. In relation to this: is there a difference in heart rate responses, either up or down, depending on how this background
vagal activity is generated: pulsatile or tetanic?
The present set of experiments was designed to tackle the
above-mentioned questions. Constant vagal restraint was induced either pulsatile (Figure 2b1) by a short burst of pulses in
each cardiac cycle systematically shifting its timing in the cycle, or tetanic (Figures 2c1 and 2c2) by applying a constant
frequency of stimulation to one vagus nerve. The sensitivity to
changes in vagal activity and the inherent delays were tested
by either increasing or decreasing the number of pulses in the
bursts or by shortly increasing the frequency or stopping the
tetanic stimulation and shifting that ‘block’ through the cardiac
cycle. Alternatively, a short burst of pulses was applied to one
vagus nerve while the other one was stimulated tetanically, so
as to mimic the effect of ‘recruitment’. Figure 2 explains the
various stimulation protocols.
These experiments had been designed to obtain qualitative
rather than quantitative results: in one and the same experiment stimulus parameters were changed to observe differences
in heart rate response in that animal. In view of the multitude
of biological and experimental variables it is a rather futile
attempt to quantify ‘the rabbit’s sinus node response’. Moreover, the experiments served to better understand human
physiology in the end.
2. Materials and Methods
Figure 2. Schematic overview of the stimulation protocols. AC = atrial
complex (indicating start of P-wave); D = variable delay between start of
P-wave and stimulus or intervention. 2a. Single burst stimulation: burst in
one cardiac cycle only at a variable delay D from start of the cycle. Number & strength of the pulses in the burst are adapted to the intended maximal amount of cycle lengthening. Responses of stimulated cycle (S) and
later (S+1, S+2, …) are referenced to the last prestimulus cycle (S-1). 2b1.
Beat-to-beat stimulation: one burst of stimuli at a variable delay D in each
cardiac cycle. The delay is kept constant until a steady state is reached.
2b2. In one particular cycle (S) the strength of the stimulus burst is
changed, either by decreasing (2b2) or increasing (2b3) the number of
pulses in the burst. 2c1. Tetanic stimulation; in one particular cycle pulses
are suppressed (2c1) or the frequency is increased (2c2) for an adjusted
period, starting at a variable delay D from the beginning of the cycle.
next - but also how much these are changed, the ensemble results of these experiments lead to so-called ‘phase response
curves’ (PRC’s) [15-17] where the induced cycle prolongation
is shown as function of the timing (phase) of the vagal burst in
the ongoing cycle (an example of such a curve is found in
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The experiments have been carried out over a period from
1979 to 1984, in accordance with prevailing law at the time
and code of ethics in animal experimentation (Declaration of
Helsinki, the UFAW handbook on the care and management of
laboratory animals [18]), under supervision of the veterinary
staff of the Jan Swammerdam Institute where the experiments
have been conducted. Fourteen New Zealand White rabbits of
both sexes have been used. Anesthesia was obtained and
maintained by intravenous injection of Nembutal (Abbott, sodium pentobarbital, initial dose up to 30 mg/kg, followed by
half the initial dose/hour as needed). In the last 9 experiments
N2O/O2 was given as inspiratory gas after anesthesia induction in combination with the pentobarbital. This resulted in
lower baseline heart rates than by pentobarbital alone. Analgesia to allow skin incisions was obtained by local infiltration
with 2% lidocaine. At the end of the experiments the animals
were euthanized by pentobarbital overdose.
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Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
la was inserted. Artificial ventilation was applied when necessary by use of Keuskamp’s ‘Amsterdam infant ventilator’
(Loosco, Amsterdam). Body temperature was maintained by a
built-in heating in the animal table. After bilateral vagotomy at
the mid-cervical level the peripheral end of the right vagus
nerve (and/or the left nerve, if necessary) was placed in a
snugly fitting flexible bipolar platinum electrode as described
elsewhere [19]. ECG was recorded from silver electrodes
placed under the skin of the thorax. A bipolar catheter mounted
electrode (4F, diameter 1.35 mm) was introduced in the right
internal jugular vein and positioned where it would detect a
well-defined atrial complex. This atrial complex was used to
trigger the timing circuits of the stimulation logic circuitry.
Electrical stimulus pulses were produced by way of a 4710
dual channel Ortec stimulator (Ortec Inc., Oak Ridge, TN,
USA), pulses were applied to each electrode by way of a Grass
S5 stimulus isolation unit (Grass Instruments, Quincy, MA,
USA). Pulse amplitudes and durations were limited to preferentially stimulate myelinated nerve fibers (max 10 V, 1.0 ms
duration). Within these limits, effectiveness of stimulation was,
as it were, titrated by varying the number of pulses within one
burst. Changing either pulse duration or voltage would change
the number of effectively stimulated nerve fibers, thereby
complicating the comparison between responses. By increasing or decreasing the number of impulses within one burst
would at least the vagal stimulation input to the sinoatrial node
be changed linearly. This is of particular importance for the
experiments described in figures 2b2 and 2b3 (beat-to-beat
stimulation with once decreased or increased burst strength).
This way of manipulating stimulation strength also helped to
limit the amount of current passed by the stimulus electrode
and to prevent damage to the nerve.
The P-wave trigger, stimulus-burst trigger and experiment
status pulses were fed directly into a DEC PDP 11/40 computer (Digital Equipment Corp., Maynard, Mass. USA) fitted with
a Lab Peripheral System, where the events were timed by a
crystal clock, at a resolution of 0.1 ms. Stimulus-response
curves were produced online. Trigger and status pulses, ECG
and atrial complex were also FM-recorded by an Ampex FR
1300 (Ampex Corp, Nivelles, Belgium) on magnetic tape as
backup. A Brush 481 polygraph pen recorder served for overview of heart rate responses.
Three stimulation protocols were followed (Figure 2).
2.1. Single burst stimulation
(Figure 2a) At an adjustable delay after a P-wave a burst of
1 to 8 pulses is given, aiming at a prolongation of around 50%
of the current cycle length; total duration of the pulse train not
exceeding 50 ms (Figures 3 and 5; Figure 4 shows as demonstration the response to a higher stimulus strength). After each
burst a rest period is programmed, enough for heart rate to
return to control value (as checked from the paper recording)
and to (manually) change the delay to scan the whole cycle.
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2.2. Beat-to-beat (‘pulsatile’) stimulation
(Figure 2b1) Each P-wave starts a chosen delay for a burst
of pulses as for single burst stimulation. Once a steady state is
reached for that delay setting, in one test cycle a programmable number of pulses from the burst is suppressed (Figure 2b2)
or extra pulses are added (Figure 2b3). The effect on that cycle
(“S”) and the following ones (S+1, …) is plotted. Then the
delay-setting is changed and the process is repeated until the
whole cardiac cycle has been scanned with different delay settings (Figures 6, 7 and 9).
2.3. Continuous (‘tetanic’) stimulation
(Figures 2c1 and 2c2) A constant stimulation frequency of
around 25 Hz is switched on. When a steady-state P-P cycle
length is reached, in a given cycle either the stimulation is
switched off for a programmable period of time (Figure 2c1)
or the frequency is increased for a short period (Figure 2c2),
while shifting that event through the cardiac cycle as in protocol 2.1 (Figure 8 and, partly, in Figure 5a, black symbols).
In the instance of fig.5a tetanic stimulation of one vagus
nerve served to manipulate P-P cycle length, while the PRC’s
were measured in response to single burst stimulation of the
other vagus nerve as in protocol 2.1. Alternatively, the P-P
cycle was shortened by single i-v dose of the ß-agonist isoproterenol (100 µg Aleudrine, Boehringer, Ingelheim).
3. Results
3.1. Single short burst stimulation
Figure 3a shows a (computer generated) ‘strip chart recording’ of the cycle lengths at the start of a stimulation series
where a burst to the right vagus nerve was scanned through the
whole cycle. In the first place the recording demonstrates that
a small respiratory sinus arrhythmia was still present, in spite
of the bilateral vagotomy. This point is discussed below, in
section 3.4. The inserted numbers at the cycle responses indicate the delay time settings in ms. They show that going from
11, 21, 31 to 51 ms in the cycle the effects on the first beat (“S”
in Figure 2a) are increasing; at 71 ms the effect already decreases and at the long delays of 151 and 171 ms there is only
an effect on the next beat (“S+1” Figure 2a), nothing in the
ongoing, stimulated cycle. Figure 3b shows this differently:
the delay of the stimulus in the cycle of the sinoatrial node is
on the X-axis. The induced cycle duration is on the Y-axis: the
blue squares for the first (stimulated) cycle, and the red triangles for the following cycle. This type of curve will be referred
to as Phase Response Curve or PRC, although it, strictly
speaking, in Figure 3b is the delay from the start of the cycle
and not the phase in the P-P cycle on the abscissa (delay divided by duration of the undisturbed cycle as in Figure 3d).
The figure shows that not all effect is lost when the stimulus
comes too late to prolong the ongoing (first) cycle: there is still
an effect on the next one. Indeed, the curves for cycle “S” and
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cycle “S+1” are, more or less, continuous when placed next to
each other, as demonstrated in Figure 3d.
Figure 3c gives the classical representation of results for
this series of stimulations. The end of each cycle after the
stimulus (which is placed for each stimulation as time = 0)
gives its point on the abscissa, the duration of that particular
cycle gives the ordinate. The latter may also be expressed in
milliseconds prolongation or as percentage of the duration of
the last pre-stimulus cycle. This way of plotting will be referred to as 'the impulse response'. Indeed, it gives the impression of an impulse response of some identifiable system;
however, it is composed of many, but all different, responses to
the same stimulus. Figure 3a also demonstrates that there is
still a, be it small, effect detectable after the first two beats.
Heart rate returned to baseline values only after 12 seconds,
when the next stimulus was applied.
In the following test in the same animal the other (left)
vagus nerve was stimulated at a higher intensity. This produced an almost ‘all or none’ response, as shown in Figures
4a-c: although I tried to get an intermediate response, as shown
by the density of data points around the transition phase in
Figure 4b this did not happen. Interestingly, many of the applied stimuli provoked a shortening of cycle duration in cycles
S+1 and/or S+2 before returning to normal, a phenomenon that
did not occur in the more moderate stimulation series as in
Figure 3.
Variability of the PRC
In the experiment of Figures 3 and 4 the basic cycle length
was changed by 2 interventions: it was increased by concurrent
tetanic stimulation of the other vagus nerve or decreased by a
single dose of isoprenaline (ß-agonist) just before the stimulation series. The resulting PRC’s are shown in Figures 5a and b.
Both give the PRC’s over the stimulated and unstimulated cycle, 5a in absolute numbers, 5b (after curve smoothing by 5
point averaging) as percentage of the control cycle, just before
the stimulation. The colors in 5a and b describe the same stimulation series: blue the shortened P-P cycle (190-200 ms), red
the baseline cycle (230-240 ms), and black the prolonged cycle
(290 ms). Figure 5b shows that the longer cycles also tend to
have longer ‘sensitive periods’ to vagal stimulation as percentage of the cycle duration. This issue is underlined by the
composition of PRC’s from 4 other experimental animals
shown in Figures 5c and d. The baseline cycle lengths were
different, due to biological variation and factors like applied
anesthesia (pentobarbital as sole anesthetic resulted in high
resting heart rates, the combination with NO2/O2 gave lower
baseline heart rates). The same line colors have been used for
series in one and the same animal. The longer resting cycles
come with definitely longer sensitive periods to vagal stimulation as percentage of the cycle duration (Figure 5d).
Figure 3. P-P cycle durations in response to a
series of 61 stimulus burst applications in one
cycle/12 seconds (burst: 4 pulses, 200 Hz, 0.3
ms duration, 9 V). 3a. P-P cycle recording of
the first 100 seconds of the series; the numbers
show the delay settings in ms for each stimulation. Note the small respiratory sinus arrhythmia. 3b. Phase response curve of the combined
results. X-axis: delay D setting (Figure 2a).
Y-axis, blue squares: resulting duration of the
first (stimulated) cycle (S); red triangles: of the
next (unstimulated) cycle (S+1). 3c. Impulse
response curve. X-axis: time between the start
of the burst and the occurrence of a P-wave for
that run. 3d. Phase response curve (%); same
data as in 3b, all data referenced to the last P-P
cycle before the stimulus (in % of
(S-1)-duration). Unstimulated second cycle
(S+1) placed next to the stimulated one (S), to
show the continuity of the phase response
curve (data 5-points averaged).
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Figure 4. Cycle durations in response to a strong stimulus (8 pulses, 200 Hz, 0.5 ms duration, 10 V) to the left vagus nerve (layout as in Figure 3). 4a:
excerpt from the cycle duration vs. time recording; numbers show the actual settings of the timing of the burst in the P-P cycle. A sharp transition between
delays 91 and 93 ms is observable. 4b: Phase response curve (%) over 2 cycles from the same data. 4c: Impulse response representation.
Figure 5. Phase response curves at different baseline P-P cycle durations (layout as in Figure 3). 5a and b are from one animal, baseline manipulated by
ß-agonist (light blue symbols) or concurrent stimulation of the other vagus nerve (black symbols). 5c and d: results from 4 different experimental animals.
Red and light blue from animals with shorter spontaneous P-P cycles, dark blue and black with longer cycles (due to change in anesthesia from pentobarbital only to the barbiturate-N2O/O2 combination). The various curves have been smoothed by a 5-point averaging filter.
3.2. Beat-to-beat stimulation
In these experiments considerably lower stimulus intensities
for the stimulus burst in each cardiac cycle had to be applied
than in the previous protocols. Only stimulus bursts could be
used that induced weak responses when applied once. Stimulus
intensities that, by themselves, would induce a strong response
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resulted in extremely low heart rates when applied repeatedly,
making blood pressure drop to values that would have made
further testing impossible. When a suitable combination of
pulse intensity, within-burst frequency and burst-duration was
found, the cycle was scanned for a number of P-to-stimulus
delay-settings, each maintained for at least half a minute.
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Two outcomes were found: one, where the shifting of the
stimulus burst through the P-P cycle had little effect on the
induced new cycle duration: it was prolonged with respect to
the resting cycle length, but the amount of prolongation was
almost independent of the timing of the vagal burst in the cycle.
The alternative outcome, at slightly higher stimulus intensities,
was one as shown in Figures 6a and b, presented as time curve
and as phase-response curve, respectively.
After a stabilization period there is an almost linear increase
of P-P cycle with increasing burst delay (Figure 6a). However,
the obtained extra cycle prolongation is slightly less than the
time shift of the stimulus. At a P-stimulus setting of 170 ms
the cycle length becomes unstable: some cycles follow the
original track and prolong further, for some the stimulation
comes too late and they are shorter (Figure 6a/b). This becomes more and more evident at longer P-stimulus delays,
until at 230 ms all cycles fall in the latter category, be it at an
increased standard deviation compared to before the unstable
region. There proved to be a definite periodicity in the jumping
up and down of the P-P cycle, i.e. it followed the period of the
ventilator (just below 2 seconds: 31 breaths/min). Both the
atrial catheter and the surface ECG showed subtle signs of
changes in atrial activation in synchrony with the P-P cycle
periodicity, which might point to shifts of the pacemaking region within the sinoatrial node (Figure 9). This issue is elaborated in section 3.4.
3.2.1. Beat-to-beat stimulation- with suddenly decreased stimulus strength
In the experiment of Figure 6a/b the stimulus in each beat
consisted of 4 pulses to the right vagus nerve. In another run in
the same experimental animal, using the same settings, the
stimulus was decreased by suppressing the first two pulses of
just one such burst. In Figure 6c the resulting cycle length is
compared to the last ‘normal’ cycle with 4 pulses, just preceding the test cycle. For the stimuli early in the cycle there is an
immediate shortening of the ongoing cycle by about 50 ms.
However, the later the burst occurs in the cycle, the more unstable this result becomes and especially in the region where
the cycle lengths become unstable (170 ms and up) the larger
Figure 6. Responses to one burst of 4 pulses in each cycle (15 ms interval, 0.2 ms duration). 6a: Overview of such an experiment; lower (red) curve denotes stepwise increased delay of the stimulus after the P-wave. Upper (black) curve shows successive P-P cycles; each dot represents one cycle. At a
delay setting of 170 ms, around 800 secs in the experiment, the cycle durations become more and more unstable at progressively longer delays. 6b: Resulting cycle durations as phase response curve; delays after the P-wave on the abscissa. 6c/d: Phase response curves to decreases of only one stimulus
burst in beat-to-beat stimulation. Layout as in 6b. 6c is from the same experiment as in fig. a/b. Blue squares give the duration of the pre-test P-P cycle. In
the test cycle the burst is halved to only 2 pulses, red triangles give the resulting duration of the test cycle. 6d is from a different experiment, same burst
stimulation settings; from the burst of 4 pulses progressively more are suppressed in the test P-P cycle, as indicated. More and more cycle shortening is
observed. (Note: adapted scales in 6d).
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Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
the shortening of the test cycle becomes. The test cycle duration can now be found at the lower ‘twig’ of the bifurcation in
Figure 6b. Finally, there is no longer a shortening effect by the
decreased burst on the ongoing beat, but the next cycle is
shortened by 35 ms. At the short P-stimulus delays the one
after the test cycle is already back to control values; data not
shown. The same pattern was found in other experiments,
where the stimulus strength was changed from 4 pulses down
to 1 pulse in the test beat. An example is shown in Figure 6d.
3.2.2. Beat-to-beat stimulation with suddenly increased stimulus strength
This experiment is, as it were, the mirror of the previous one,
and the results can very well be interpreted as such. Figure 7a
shows the case where 2 pulses/cycle had a slight prolonging
effect on the P-P cycle, almost independent of the timing in the
cycle. Addition of 6 pulses once to such a burst exposed the
underlying PRC: early prolonged bursts would increase the
P-P cycle from around 400 to 460 ms; late extra pulses had no
197
effect on the ongoing cycle and only a minor effect on the next
one. In the alternate case, shown in Figure 7b the bursts were
increased from 6 to 8 pulses in the test cycle. The PRC was
already apparent in the ‘background’ effect induced by 6 pulses/cycle; addition of 2 extra pulses worked only for the early
bursts in the ongoing cycle, with some remnant effect on the
next cycle for late bursts. However, in the transition phase,
where the effect of 6 pulses was unstable, the addition of 2
extra pulses had a large effect on the ongoing cycle and the
next one.
3.3. Continuous (‘tetanic’) stimulation
This type of vagus nerve stimulation has been studied extensively in the literature. Rather unexpectedly I found very variable heart rates from one beat to the next, even though the
stimulation frequency was held constant. In the present experiments only the effects of short-lasting increased or decreased frequency blocks was studied. Compared to the previous - pulsatile - modes of stimulation it was striking how much
Figure 7. Responses to increases of only one stimulus burst in beat-to-beat stimulation. Drawn red lines give the undisturbed interval durations on
beat-to-beat stimulation. 7a: bursts of 2 stimuli, in the test interval increased to 8. Squares: duration of test interval, triangles: next interval. 7b: bursts of 6
stimuli, also increased to 8. Resting heart period around 330 ms.
more/longer the test blocks had to be to provoke heart rate
effects that would clearly stand out of the background variability. The issue of ‘vagal noise’ is elaborated below in section
3.4.
3.3.1. Tetanic stimulation with short suppression of stimuli
Figure 8a/b shows an example of such an experiment: tetanic stimulation at 25 Hz had increased cycle length from a
resting 290 ms to around 520 ms. Suppression of stimulation
for 400 ms led to a maximal shortening effect to around 400
ms (Figure 8a), obtained in the second cycle after the suppression of stimuli had started (Figure 8b). The PRC for cycles 1
and 2 demonstrate that only a small effect was reached in the
first cycle, if stimuli had been stopped in the first 200-250 ms;
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ond cycle. The largest effects occurred in the second cycle for
those instances where the full block of stimuli had occurred
just before the end of the first which it had left uninfluenced.
The P-P cycle was quickly back to earlier values, even slightly
overshooting those for a short period of time.
3.3.2. Tetanic stimulation with short-lasting frequency increase
This experiment should come out more or less the same as
the one described in Figure 5a/b (black symbols), where on
one vagus nerve the tetanic frequency was applied, while the
other nerve served to apply a short-lasting extra stimulus. Here
one and the same nerve and same stimulus intensity settings
were used to, shortly, increase the frequency. This encountered
the same problems as mentioned under 3.3.1: a sizeable
change had to be applied to induce a response that would
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clearly stand out of the background noise. Since this protocol
also implied intense use of the same vagus fibers over and over,
fading of the response was observed in the course of a test run
[20]. Therefore, the result in Figure 8c/d is an average of 2
such runs: one where the delay setting was going up and the
other where it was going down (from 10 to 320 ms and reverse). The baseline cycle of 260 ms was prolonged by the
tetanic stimulation to 340 ms. Both the impulse response curve
and the PRC look like the ones shown in section 3.1, with one
exception: depending on the circumstances the apparent delay
of the impulse may turn out much longer than the value of
around 100-120 ms that can be read from Figure 3, rather more
like 200 ms.
some extra background variability, above and beyond the biological variation that occurs anyway.
The situation is, however, more complicated: depending on
the applied stimulus mode, the observed RSA was amplified or
dampened. In the beat-to-beat stimulation (protocol 2) it, generally, was amplified, extremely so when the stimulus was
applied in the ‘unstable’ region (Figure 6 and Figure 9, below);
under tetanic stimulation (protocol 3) it was not always observed, or it sort-of drowned in a high-frequency background
noise of fast beat-to-beat variability without a defined predilection frequency, as confirmed by frequency analysis of the
time series (results not shown).
3.4. Unsolicited results
The main objective of this study was to investigate how the
physiological properties of the vagus nerve-sinoatrial node
complex in vivo translate changes in cardiac vagal activity into
heart rate variability. To do this, electrical stimulation of the
efferent vagus nerves was applied after bilateral vagotomy. As
shown in the results section, the heart rate effects of (changes
In the present experiments, where both vagi have been cut,
one would expect a stable baseline of heart rate, devoid of the
fast beat-to-beat variations, if these were only vagally
mediated. Rabbits are known to make an exception to that rule:
even after bilateral vagotomy a small respiratory sinus arrhythmia (RSA) of a few milliseconds peak-to-peak can still
be observed [21]. In all result curves shown above this caused
4. Discussion
Figure 8. Responses to short lasting decreases or increases of tetanic stimulation at 25 Hz (layout as in Figure 2a/b). 8a/b: suppression of stimulation for
400 ms. 8c/d: frequency increased to 100 Hz for 240 ms. The dots in 8c/d are averages of 2 runs: one with increasing and one with decreasing ‘stimulus’
delays.
Distributed under creative commons license 4.0
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Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
Figure 9. Stretch of the recording from the experiment with beat-to-beat
stimulation in fig. 6, delay-setting of the stimulus in the cycle ~ 170 ms.
Top: surface ECG, AC: atrial complex from catheter electrode, below:
trigger pulse started by atrial depolarization, schematic indication of 4
stimuli. The stimulus artefacts are clearly visible in ECG and AC. The
cycle duration jumps between ~ 360 and 550 ms. The arrows point to
slightly changed AC’s ending the longer cycles, possibly signs of pacemaker shifts.
in) vagal activity depend strongly on the timing of this activity
in the sinoatrial node cycle and on the prevailing conditions.
An overwhelming amount of literature has laid a solid basis
for understanding of how the parasympathetic system
influences the functioning of the cardiovascular system, dating
back to 1868 with the work of Donders, who for the first time
called attention to the phase dependency of the heart rate effect
of vagal stimulation [15,17,22-30]. In many of those studies
this effect and those of different stimulation frequencies have
been investigated, however, the issue of temporary increase or
decrease of stimulus intensity has received very little attention.
It may seem that this issue is covered by modulation of the
frequency of vagus nerve stimulation, going up and down following a sinusoidal pattern. When sinusoids of a broad range
of frequencies are used, one may assume to have caught all
dynamic aspects of the relation between vagus activity and
sinoatrial node response (“transfer function analysis”), as it has
been applied by a number of authors [31-34]. Of these the experiments by [35] come closest to what has been presented
here: these authors applied stimulation coupled to the P-wave,
which was slowly frequency modulated, modulation frequency
spanning at least several heartbeats. However, that type of
system analysis supposes a more or less linear system, not one
where the timing of a stimulus may induce different effects,
depending on its arrival time in the cardiac cycle. This relates
to a basic issue that is haunting the field of HRV: heart beats,
or rather R-R-intervals, are what is being measured on a
beat-to-beat scale which has its own dynamic, due to processes
like the ones studied in this paper. Therefore, newly derived
numbers like sample entropy or multiscale entropy are using
the original R-R-interval series [36,37]. In some other analysis
methods, it has become customary to interpolate the RR-values
to have a signal that is regularly sampled at fixed intervals
[32,33,35]. This serves to solve the problem that most processes also have a time-dependent component, and representaDistributed under creative commons license 4.0
199
tions of HRV where the timescale is not readily apparent, cannot easily be interpreted. This becomes an even more pressing
problem if there are substantial changes of the prevailing heart
rate in the course of the observation period. Interpolation of
the original time series by whichever method [38-40] cannot
redress this problem. As is shown in this study, low heart rate
will show dynamics different from that at higher heart rates, be
it alone by the changed PRC and the timing of vagal bursts in
the cycle.
All through this study the Phase Response Curve has been
shown to be of paramount importance to understand the effect
of a cardiac vagal impulse on heart rate. Also that observation
is not new; PRC’s have often been studied, e.g. in [17,30,41],
however, the application of repeated, short-lasting stimulus
bursts was different from the approach chosen here: those authors used the timing of the bursts as the driving mechanism,
independent of the beat occurrences, to find regions of ‘entrainment’ (e.g. [14,16]) where HR would follow that timing.
One might think of vagal bursts induced by expiration as a
more or less independent mechanism to deliver such a driving
signal. However, respiration rate is generally (much) lower
than heart rate and lower than the applied rate of vagal burst
stimulation in those entrainment experiments. In vivo, roles
are reversed: the vagal bursts follow each heartbeat, as they are
locked to the pulse wave upstroke by way of the baroreflex.
In Figures 3 and 4 it was demonstrated that the heart rate
reaction to a vagal burst has two components: 1) an immediate,
strong effect, lasting at most one beat and the next and 2) a
slow after-effect that may last for several seconds. The immediate response is phase-sensitive; a burst coming too late in the
ongoing cycle may at most have a diminished effect on the
next one. The slow after-effect is not as phase-sensitive as that
and it is only small, compared to the immediate effect. However, it builds up with repeated stimulation and requires many
seconds to disappear. That may be why, in the case of tetanic
stimulation, it is so difficult to induce an immediate, sizeable
cycle shortening, and even with prolonged suppression of the
ongoing stimulation cycle length does not fall back to the unstimulated value (Figure 8a/b).
Beat-to-beat stimulation early in the cycle gave the largest
and most stable responses; when the stimulation occurred in
the region where the response became unstable, a small disturbance like that caused by respiration (be it artificial or
spontaneous ventilation) could impose its rhythm on the unstable situation, consequently inducing a large RSA in the face
of an unchanging vagal stimulus to the heart (Figure 6). To my
knowledge this has not been described in literature before.
When the stimulus came late in the cycle, a more or less stable
effect on the following cycle was observed, less effective
though than stimulation early in the cycle (but still with a sizable RSA).
The above does not explain the results of tetanic
low-frequency stimulation. Since the pulses were not locked to
the P-wave, there was variability in where and how many
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Karemaker | Journal of Clinical and Translational Research 2015;1(3): 190-204
pulses would occur in the sensitive period of the prevailing
PRC, later coming pulses in that cycle are ineffective. This
process may be expected to lead to some variability in cycle
times. Additionally, the transfer in the cardiac ganglia is probably not of the one-to-one type; since the ganglions cells are
known to display autonomous subthreshold oscillations and
spontaneous activity [42], interference with the imposed stimulation frequency might act as an additional cause of variability. Most of the cycle prolongation under this type of stimulation is probably due to what was called above the ‘built-up
after-effect’. Consequently, only when the suppression of
stimulation starts very early (and inhibits a sizeable number of
pulses) would the first cycle be shortened (Figure 8a/b), the
largest effect to occur in the next cycle. The opposite: a
short-lasting block of increased frequency behaves just like the
impulse responses described in the beginning, now against the
background of an increased cycle length (Figure 8c/d).
4.1. The baroreflex, hrv and timing of vagal bursts
This study provides more insight into the working of the
baroreflex on heart rate. One may now ask, how important is
this PRC to the responsiveness of the sinoatrial node to its in
vivo vagal input. In other words, in particular when looking at
Figure 6, what does this mean for the normally arriving vagal
burst after a ventricular contraction? In the experiments stimulation was coupled to the atrial complex, since the heart rate is
generated in the atrium, and coupling to the (ventricular)
R-wave is inherently biased by possible timing differences due
to the vagal (and other -) effects on AV-conduction. Figure 9
shows the surface ECG and atrial complex during repeated
stimulus bursts ~170 ms after the P-wave, i.e. in the unstable
phase of the sinoatrial node cycle. The AV-conduction is
slightly prolonged, the vagal burst occurs around the T-wave,
i.e. some 80-100 ms after ventricular contraction has started.
This is around the very first moment that a vagal response to
baroreceptor afferent activity might have been expected in
rabbits, if we assume the estimates of reflex latency in the cat
to be applicable to rabbits as well [10,12,43], where values of
60-70 ms, 26-90 ms and 20-60 and/or 70-110 ms respectively
were found. The latency of 100-120 ms which I found in my
thesis work in awake rabbits from HR responses to carotid
sinus nerve and depressor nerve stimulation, is slightly longer
[44]. In our experiments in humans [45] we estimated still
longer reflex times, extrapolating from electrical stimulation of
carotid sinus nerves to heart rate responses (0.35 s). However,
in view of a number of uncertainties in those measurements,
which have later been stressed by Eckberg and coworkers [46]
this estimate might be over-cautious and therefore too long.
Therefore, although these are experiments in rabbits, the
results have relevance for baroreflex functioning and related
HRV in human physiology. This holds in particular for the
regulation at quiet heart rates, below 75 beats/min [47], where
changes in systolic pressure in the ongoing beat are known to
influence the occurrence of the upcoming P-wave. A more
Distributed under creative commons license 4.0
elaborate discussion of this issue has been given in [48] and
[45]. In the computation of baroreflex sensitivity (BRS) the
original algorithm is to correlate the systolic pressures in the
rising phase of a phenylephrine induced pressure rise to the
durations of the succeeding beats [49]. At heart rates lower
than 75 this should be the duration of the very beat where the
systolic pressure was measured. From the experiments presented here one may infer that the heart rate lowering is even
more effective if the vagal pulses arrive still earlier in the cycle,
therefore at substantially lower heart rates than 75/min. HRV is
known to diminish at higher HR, this is probably due to the
vagal pulses coming too late for the ongoing beat to have their
full effect, so it is delayed –with decrement- to the next beat.
This also explains the increasing beat delay observed at higher
heart rates in the systolic pressure to heart period correlation
such as used in the running baroreflex sensitivity xBRS [50] or
the phase delay between systolic pressure and heart period
changes in Fourier analysis [1]. This delay need not necessarily be a sign of increasing sympathetic involvement in the baroreflex to heart rate response. Since this effect of diminishing
response to the same vagal burst activity does not depend on
the frequency of some underlying oscillation, be it respiratory
(high frequency or “HF”) or sympathetically mediated
10-seconds oscillation (low frequency or “LF”), one cannot
predict from this how a change in HR might affect a quotient
like LF/HF [51]. Other typically vagal measures, like RSA or
BRS, will definitely give lower numbers.
4.2. Respiratory sinus arrhythmia and the vagus nerve
As was shown above, even after bilateral vagotomy RSA
was observed. In the literature this phenomenon has been attributed to direct stretch of the sinoatrial node [21,52]. It might
equally well be due to cardiac stretch receptors synapsing with
efferent autonomic nerves in the ganglionic plexus near the
heart [7,53]. In the present experiments RSA increased dramatically when the vagus was stimulated at a critical timing in
the cycle of the sinus node (Figures 6 and 7). Whatever the
exact cause of RSA in the resting situation, from physics we
know that a minute disturbance may tip the balance in an unstable system; respiratory movement might be just that disturbance. This might also be an explanation for the extreme
RSA that is sometimes observed in young persons, where HR
may jump from around 60 to 100 bpm and back in a few beats
(personal observation).
4.3. The sinus node under vagal control
All along this paper has been about the originator of heart
rate, the sinus node, and how it reacts to vagal influences. A
number of basic issues related to known vagus nerve and sinoatrial node function should still be discussed. First of all, the
delay time between arrival of acetylcholine at the end organ
and the first measurable change of function in that organ. It is
well-known that this delay after activation of the (postganglionic) muscarinic receptor is much longer than that at the
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Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
nicotinic receptor (like the ones in the ganglia and the neuromuscular junction). The latter one has delay times in the order
of 0.2 ms, the former in the order of 100-150 ms [54,55]. This
implies that the shortest latency between vagus nerve stimulation and shifting the next P-wave to a later moment is very
close to this ‘muscarinic transmission delay’ in the present
experiments (Figure 3). Second, which phase of the sinoatrial
node action potential should be ‘hit’ by vagal pulses to obtain
most effect? I was given the opportunity by Drs. L.N. Bouman
and F.I.M. Bonke to re-analyze an archived copy of their experiments on vagus nerve stimulation in isolated rabbit sinoatrial node preparations [56]. They measured the sinoatrial
node response to stimulation by trains of 25 Hz lasting a few
seconds. The responses in these rabbits were unlike those in
Jalife and Moe’s experiments in a comparable preparation in
young cats [57]. The latter authors applied strong stimulus
bursts, to which the sinoatrial node would react by a full ‘reset’, i.e. the action potential was pushed from its depolarization course back to the maximal hyperpolarization level and
had to restart from the lowest point (Figure 1 more or less depicts this situation). The starts of a tetanic stimulation by
Bouman and Bonke were like the one demonstrated in Figure
10, where no reset but a gradual deviation from the unstimulated course would show.
Comparing the occurrence of the atrial complex in Figure
10 to the PRC’s observed in my own experiments (started by
the atrial depolarization as well) the most effective timing of a
single stimulation burst is in the late repolarization-early diastolic phase of the sinus node action potential. This is in good
agreement with Jalife and Moe’s [57] measurements.
Figure 10. Recording from an experiment by L.N. Bouman and F.I.M.
Bonke: micro-electrode in rabbit sinoatrial node cell (SN) while the attached vagus nerves were stimulated. AC: atrial complex, recorded from
the crista terminalis. At the rightmost up-arrow 25 Hz vagus nerve stimulation started. The last unstimulated and first stimulated cycles have been
superimposed (repeated up-arrow to the left). Stimulation is evident from
the large artefacts. At the down-arrow the first change from unstimulated
to stimulated action potential is detectable. The earlier this occurs in the
cycle, the larger the effect on the ongoing depolarization. For clarity the
last unstimulated cycle has been touched up in red from that point onward.
Figure has been published earlier in [44] as Figure 3.2.
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201
Finally, the short- vs. the long-lasting effects of vagal stimulation; is there a basis in known sinoatrial node properties?
An extensive review of the literature has been published in [58]
ranging from the various types of muscarinic receptors in sinoatrial node tissue to the membrane channels involved in the
translation of acetylcholine effect to changed membrane channel properties. It may be concluded that the hypothesis formulated above has a sufficiently sound basis in the known properties of the sinoatrial node, i.e. vagal influences may be distinguished in fast and short-lasting, explaining the sudden prolongations of cycle length, vs. slowly building up effects of
repeated stimulation and the relatively slow return to the baseline values afterwards. Only the fast influence on HR of a vagal burst can be quickly undone from one heartbeat to the next,
the slow response takes several beats to disappear.
4.4. HRV and the vagus nerve
The cardiac vagus nerve fibers, serving as efferent pathway
to the baroreflex, provide immediate adaptation of heart rate to
changes in blood pressure. Therefore, the efferent cardiac vagal bursts, one per heartbeat, might be considered translations
of the incoming baroreceptor afferent bursts that are caused by
pulsatile stretch of the vessel wall at each heartbeat. However,
this conversion is not one-to-one: already in the first transmission station, the nucleus tractus solitarii of the medulla oblongata, integration takes place with other incoming visceral and
somatic afference [10,43]. In addition, the central interaction
with respiration induces a partial blockade of vagal outflow
during the inspiratory phase [10,43,59]. Consequently, the
outgoing cardiac vagal traffic is not only a reflex response to
the baroreceptive input signals. Moreover, the ganglionic cardiac plexus is not the simple textbook transmission station;
ganglion cells themselves show subthreshold oscillations and,
on top of that, incoming neural traffic from cardiac sensory
neurons may alter their activity [7].
The combined effects of heart rate on the effectiveness of
incoming vagal traffic, i.e. the change in duration of the ‘sensitive period (shorter at higher HR) and the relatively late arrival of vagal bursts are good explanations for the observed relationship between HRV and HR, mentioned in the introduction
[4,5]. This does not settle the ‘hot’ issue whether it is better to
take heart period or heart rate for the calculations of HRV.
Parker et al. showed in 1984 [60] a nice linear correlation between amount of vagal activity (stimulus pulses) and obtained
heart period, an observation that underlined Jewett’s earlier
finding that the number of preceding cardiac vagal impulses
correlated well with the length of the heart period [11]. These
observations have given credence to the corollary that heart
periods should best be measured to account for vagal effects
(mainly in the resting condition) and heart rates during exercise, looking at the effect of the sympathetics. When looking at
the 1/x relationship between the two, one may also argue that
in the resting condition heart period changes would almost
vanish if expressed as HR-changes, and vice versa. A more
elaborate discussion of the issue has been given earlier [61].
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The issue raised by Yaniv et al. [6,62] relating to inherent
stochastic behavior of the sinoatrial node as cardiac pacemaker
is yet another matter. Pacemaker cells, when isolated in tissue
culture, display widely varying intervals between firings [63].
Once the cells have replicated and form a syncytial-like tissue,
the interval becomes stabilized. In vivo it is a known fact that
cells in the pacemaking region may show different behaviors:
from ‘true pacemaking cells’ where the earliest activity in the
cycle can be recorded, to ‘latent pacemaking cells’ which display a slower diastolic depolarization and a sharp transition to
the systolic upstroke [64,65]. The postganglionic sympathetic
and vagal fibers have an uneven distribution over that area,
consequently the actual pacemaking region may shift with
autonomic activity [56]. An example of that may be observed
in Figure 9. Even more extreme might the sinoatrial node be
blocked to such an extent, that a vagal escape occurs and the
actual pacemaker shifts to another pacemaking area, like the
AV-node, for one or more beats. That did not occur in the present experiments, where care was taken not to go to extremes
with vagal stimulation, but the phenomenon of vagal escape is
well-known in physiological literature [66,67] and, of course,
from clinical observations like in ophthalmic surgery [68]
where manipulation of the eye can produce strong sinoatrial
node inhibition, and the AV-node temporarily may take over
pacemaking of the heart.
5. Conclusions
In this series of experiments, the importance of the
Phase-Response Curve for understanding vagally induced
phasic heart rate changes has been established, both for single
burst and for beat-to-beat burst-like stimulation. The latter can
be considered the normal in-vivo mode of operation in response to baroreceptor input due to the pulse wave upstroke,
making vagal activity the most important contributor to HRV
in the healthy, supine resting condition. The experiments have
also shown that the PRC is not a constant given, but a changing operator, depending on the prevailing condition of the sinoatrial node and immediately prior vagus nerve activity. Under conditions the response to a vagal burst may become unpredictable, when its timing in the cycle has become critical. If
vagus nerve activity does not come beat-to-beat, but as a more
or less continuous background ‘vagal tone’, it induces slower
on- and off-responses than those provoked by burst-like activity.
In general, the HR-response to vagal activity shows two
components: a fast one that may provoke large cycle changes
from one beat to the next, and a slow one, that builds up with
longer lasting activity. The latter one also takes longer to subside, in small steps.
The response to vagal stimulation is not only determined by
the properties of primary pacemaking cells in the sinoatrial
node, but also by the properties of the ganglion cells in the
cardiac plexus and by the unequal distribution of their
end-organ effects, leading to pacemaker shifts within the sinoatrial node or even to other areas, like the AV-node. The exisDistributed under creative commons license 4.0
tence of RSA, even after vagotomy, may be explained by integrating action of the cardiac autonomic plexus where vagal,
sympathetic and cardio-sensory information come together.
6. Limitations
Blood pressure decrease during vagal stimulation may have
induced increased sympathetic drive to the sinoatrial node
(However, control experiments after ß-blockade by propranolol did not give remarkably different results). The change in
applied anesthesia in the course of the experimental series,
from barbiturate-only to barbiturate and N2O/O2, in order to
have animals with lower baseline heart rates, may have induced more variable heart rates at the same time.
Acknowledgements
In the course of the experiments, stretched out over the
years, many colleagues have helped directing the experimental
set-up to its final shape, presented here. The technical support
of Ms. J. Veltman and, later, Mr. W.J. Stok has been essential.
Disclosures
The author has no relevant disclosures to make. The experiments have been carried out in the former Dept. of Physiology at the Medical Faculty of the University of Amsterdam,
where he worked as associate professor of Physiology.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
DeBoer RW, Karemaker JM, Strackee J. Hemodynamic fluctuations and baroreflex sensitivity in humans: a beat-to-beat
model. Am J Physiol. 1987; 253: H680-H9.
Karemaker JM, Wesseling KH. Variability in cardiovascular
control: the baroreflex reconsidered. Cardiovascular Engineering. 2008; 8: 23-9.
Karemaker JM. Counterpoint: Respiratory sinus arrhythmia is
due to the baroreflex mechanism. Journal of applied physiology.
2009; 106: 1742-3.
Billman GE. The effect of heart rate on the heart rate variability
response to autonomic interventions. Frontiers in Physiology.
2013; 4: 222.
Monfredi O, Lyashkov AE, Johnsen A-B, Inada S, Schneider H,
Wang R, Nirmalan M, Wisloff U, Maltsev VA, Lakatta EG,
Zhang H, Boyett MR. Biophysical characterization of the underappreciated and important relationship between heart rate
variability and heart rate. Hypertension. 2014; 64: 1334-43.
Yaniv Y, Tsutsui K, Lakatta E. Potential effects of intrinsic heart
pacemaker cell mechanisms on dysrhythmic cardiac action potential firing. Frontiers in Physiology. 2015; 6.
Armour JA. Potential clinical relevance of the ‘little brain' on
the mammalian heart. Experimental Physiology. 2008; 93:
165-76.
Karemaker J. Chaos in heart rate and the vagus nerve. A theoretical and experimental investigation. 3rd International workshop on computer analysis of blood pressure and heart rate sig-
DOI: http://dx.doi.org/10.18053/jctres.201503.005
Karemaker | Journal of Clinical and Translational Research 2015; 1(3): 190-204
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
nals; 4-5 May 1995; Firenze, Italy1995.
Karemaker JM. Vagal effects on heart rate: Different between
up and down. Cardiovascular Oscillations (ESGCO), 2014 8th
Conference of the European Study Group on; 2014 25-28 May
2014.
Iriuchijima J, Kumada M. Activity of single vagal fibers efferent
to the heart. The Japanese Journal of Physiology. 1964; 14:
479-87.
Jewett DL. Activity of single efferent fibers in the cervical vagus
nerve of the dog, with special reference to possible cardio-inhibitory fibres. The Journal of Physiology. 1964; 175: 321-57.
Kunze DL. Reflex discharge patterns of cardiac vagal efferent
fibres. J Physiol (London). 1972; 222: 1-15.
Levy MN, Martin PJ. Neural regulation of the heartbeat. Ann
Rev Physiol. 1981; 43: 443-53.
Guevara MR, Glass L. Phase locking, period doubling bifurcations and chaos in a mathematical model of a periodically driven oscillator: A theory for the entrainment of biological oscillators and the generation of cardiac dysrhythmias. J Math Biol.
1982; 14: 1-23.
Levy MN, Martin PJ, Iano T, Zieske H. Paradoxical effect of
vagus nerve stimulation on heart rate in dogs. Circ Res. 1969;
25: 303-14.
Michaels DC, Slenter V, A. J., Salata JJ, Jalife J. A model of
dynamic vagus-sinoatrial node interactions. Am J Physiol. 1983;
245: H1043-H53.
Abramovich-Sivan S, Akselrod S. A phase response curve
based model: effect of vagal and sympathetic stimulation and
interaction on a pacemaker cell. J Theo Biol. 1998; 192:
567-79.
UFAW Staff. The UFAW handbook on the care and management of laboratory animals. 3rd edition. London and Edinburgh:
E. & S. Livingstone; 1967.
Karemaker JM, Borst C, Schreurs AW. Implantable stimulating
electrode for baroreceptor afferent nerves in rabbits. Am J
Physiol. 1980; 239: H706-9.
Salata JJ, Jalife J. "Fade" of hyperpolarizing responses to vagal
stimulation at the sinoatrial and atrioventricular nodes of the
rabbit heart. Circ Res. 1985; 56: 718-27.
Perlini S, Solda PL, Piepoli M, Sala-Gallini G, Calciati A, Finardi G, Bernardi L. Determinants of respiratory sinus arrhythmia in the vagotomized rabbit. Am J Physiol. 1995; 269:
H909-15.
Donders FC. Zur Physiologie des Nervus Vagus. Pflügers Archiv ges Physiologie. 1868; 1: 331-61.
Brown GL, Eccles JC. The action of a single volley on the
rhythm of the heart. J Physiol (London). 1934; 82: 211-41.
Brown GL, Eccles JC. Further experiments on vagal inhibition
of the heartbeat. J Physiol (London). 1934; 82: 242-57.
Levy MN, Martin PJ, Iano T, Zieske H. Effects of single vagal
stimuli on heart rate and atrioventricular conduction. Am J
Physiol. 1970; 218: 1256-62.
Stuesse SL, Levy MN, Zieske H. Phase-related sensitivity of
the sinoatrial node to vagal stimuli in the isolated rat atrium.
Circ Res. 1978; 43: 217-24.
Distributed under creative commons license 4.0
203
[27] Levy MN, Wexberg S, Eckel C, Zieske H. The effect of changing interpulse intervals on the negative chronotropic response to
repetitive bursts of vagal stimuli in the dog. Circ Res. 1978; 43:
570-6.
[28] Dong E, Reitz BA. Effect of timing of vagal stimulation on
heart rate in the dog. Circ Res. 1970; 27: 635-46.
[29] Levy MN, Lano T, Zieske H. Effects of repetitive bursts of
vagal activity on heart rate. Circ Res. 1972; 30: 186-95.
[30] Jalife J, Slenter VA, Salata JJ, Michaels DC. Dynamic vagal
control of pacemaker activity in the mammalian sinoatrial node.
Circ Res. 1983; 52: 642-56.
[31] Peňáz J. Frequency response of the cardiac chronotropic action
of the vagus in the rabbit. Arch Physiol Biochem. 1962; 70:
636-50.
[32] Berger RD, Saul JP, Cohen RJ. Transfer function analysis of
autonomic regulation. I. Canine atrial rate response. Am J
Physiol. 1989; 256: H142-52.
[33] Saul JP, Berger RD, Chen MH, Cohen RJ. Transfer function
analysis of autonomic regulation. II. Respiratory sinus arrhythmia. Am J Physiol. 1989; 256: H153-61.
[34] Saul JP. Transfer function analysis of cardiorespiratory variability to assess autonomic regulation. Clin Sci (Colch). 1996; 91
Suppl: 101.
[35] Mokrane A, LeBlanc AR, Nadeau R. Transfer function analysis
of vagal control of heart rate during synchronized vagal stimulation. Am J Physiol. 1995; 269: H1931-40.
[36] Richman JS, Moorman JR. Physiological time-series analysis
using approximate entropy and sample entropy. Am J Physiol
Heart Circ Physiol. 2000; 278: H2039-49.
[37] Costa M, Goldberger AL, Peng CK. Multiscale Entropy Analysis of Complex Physiologic Time Series. Phys Rev Lett. 2002;
89: 068102.
[38] de Boer RW, Karemaker JM, Strackee J. Description of
heart-rate variability data in accordance with a physiological
model for the genesis of heartbeats. Psychophysiology. 1985;
22: 147-55.
[39] Berger RD, Akselrod S, Gordon D, Cohen RJ. An efficient algorithm for spectral analysis of heart rate variability. IEEE
Trans Biomed Eng. 1986; 33: 900-4.
[40] Camm AJ, Malik M, Bigger JT, Breithardt G, Cerutti S, Cohen
RJ, et al. Heart Rate Variability - Standards of Measurement,
Physiological Interpretation, and Clinical Use [Review]. Circulation. 1996; 93: 1043-65.
[41] Slenter VA, Salata JJ, Jalife J. Vagal control of pacemaker periodicity and intranodal conduction in the rabbit sinoatrial node.
Circ Res. 1984; 54: 436-46.
[42] McAllen RM, Salo LM, Paton JFR, Pickering AE. Processing
of central and reflex vagal drives by rat cardiac ganglion neurones: an intracellular analysis. J Physiol. 2011; 589: 5801-18.
[43] McAllen RM, Spyer KM. The baroreceptor input to cardiac
vagal motoneurones. J Physiol. 1978; 282: 365.
[44] Karemaker JM. Vagal effects of the baroreflex on heart rate
[PhD thesis]. Amsterdam: University of Amsterdam; 1980.
[45] Borst C, Karemaker JM. Time delays in the human baroreceptor reflex. J Auton Nerv Syst. 1983; 9: 399-409.
DOI: http://dx.doi.org/10.18053/jctres.201503.005
204
Karemaker | Journal of Clinical and Translational Research 2015;1(3): 190-204
[46] Seidel H, Herzel H, Eckberg DL. Phase dependencies of the
human baroreceptor reflex. Am J Physiol Heart Circ Physiol.
1997; 41: H2040-H53.
[47] Pickering TG, Davies J. Estimation of the conduction time of
the baroreceptor-cardiac reflex in man. Cardiovasc Res. 1973; 7:
213-9.
[48] Karemaker JM, Borst C. Measurement of baroreflex sensitivity
in hypertension research. In: Sleight P (editor): Arterial baroreceptors and hypertension. Oxford University Press: Oxford;
1980. p. 455-61.
[49] Smyth HS, Sleight P, Pickering GW. Reflex regulation of arterial pressure during sleep in man. A quantitative method of assessing baroreflex sensitivity. Circ Res. 1969; 24: 109-21.
[50] Westerhof BE, Gisolf J, Stok WJ, Wesseling KH, Karemaker
JM. Time-domain cross-correlation baroreflex sensitivity: performance on the EUROBAVAR data set. J Hypertens. 2004; 22:
1371-80.
[51] Force T. Heart rate variability. Standards of measurement,
physiological interpretation, and clinical use. Task force of the
European society of cardiology and the North American society
of pacing and electrophysiology. Eur Heart J. 1996; 17: 354-81.
[52] Golenhofen K, Lippross H. Mechanishce Koppelungswirkungen der Atmung auf den Herzschlag [Mechanical coupling effects between respiration and heart rhythm]. [German]. Pflügers
Arch. 1969; 309: 159-66.
[53] Bolter CPWSJ. Influence of right atrial pressure on the cardiac
pacemaker response to vagal stimulation. Am J Physiol Regul
Integr Comp Physiol. 1999; 276: R1112-R7.
[54] Purves RD. Muscarinic excitation: a microelectrophoretic study
on cultured smooth muscle cells. Br J Pharmacol. 1974; 52:
77-86.
[55] Purves RD. Function of muscarinic and nicotinic acetylcholine
receptors. Nature. 1976; 261(13 May 1976): 149-51.
[56] Bouman LN, Gerlings ED, Biersteker PA, Bonke FIM. Pacemaker shift in the sino-atrial node during vagal stimulation.
Pfluegers Arch. 1968; 302: 255-67.
[57] Jalife J, Moe GK. Phasic effects of vagal stimulation on pace-
Distributed under creative commons license 4.0
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
maker activity of the isolated sinus node of the young cat. Circ
Res. 1979; 45: 595-608.
Demir SS, Clark JW, Giles WR. Parasympathetic modulation of
sinoatrial node pacemaker activity in rabbit heart: a unifying
model. Am J Physiol Heart Circ Physiol. 1999; 276: H2221-44.
Gilbey MP, Jordan D, Richter DW, Spyer KM. Synaptic mechanisms involved in the inspiratory modulation of vagal cardio-inhibitory neurones in the cat. J Physiol. 1984; 356: 65-78.
Parker P, Celler BG, Potter EK, McCloskey DI. Vagal stimulation and cardiac slowing. J Auton Nerv Syst. 1984; 11: 226-31.
Faes TJ, De Neeling NN, Kingma R, TenVoorde BJ, Karemaker
JM. On the quantification of heart rate changes in autonomic
function tests: relations between measures in beats per minute,
seconds and dimensionless ratios. Clin Sci (Colch). 1995; 89:
557-64.
Yaniv Y, Lyashkov AE, Lakatta EG. Impaired signaling intrinsic to sinoatrial node pacemaker cells affects heart rate variability during cardiac disease. J Clin Trials. 2013; 4: 152.
Jongsma HJ, Tsjernina L, de Bruijne J. The establishment of
regular beating in populations of pacemaker heart cells. A study
with tissue-cultured rat heart cells. J Mol Cell Cardiol. 1983; 15:
123-33.
Lu H-H, Lange G, McC. Brooks C. Factors Controlling Pacemaker Action in Cells of the Sinoatrial Node. Circ Res. 1965;
17: 460-71.
Bouman LN, Jongsma HJ. Structure and function of the sino-atrial node: a review. Eur Heart J. 1986; 7: 94-104.
Hill L, Barnard H. The Influence of the Force of Gravity on the
Circulation Part II. Section I. The action of the respiratory
pump. Section II. The escape of the heart from vagal arrest.
Section III. The mean pressure of the vascular system. J Physiol.
1897; 21: 323-52.
Wallace AG, Daggett WM. Pacemaker activity during vagal
escape rhythms. Circ Res. 1964; 15: 93-102.
Donlon JV Jr. Anesthesia for ophthalmologic surgery. ASA
Refresher Courses in Anesthesiology. 1988; 16: 81-92.
DOI: http://dx.doi.org/10.18053/jctres.201503.005